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. 1989 Mar;410:325–339. doi: 10.1113/jphysiol.1989.sp017535

Expression of amino acid transport systems in cultured human umbilical vein endothelial cells.

G E Mann 1, J D Pearson 1, C J Sheriff 1, V J Toothill 1
PMCID: PMC1190481  PMID: 2677320

Abstract

1. Nutrient transport in cultured human umbilical vein endothelial cells was characterized using a rapid dual-isotope dilution technique. Microcarrier beads with confluent endothelial cells were perfused in small columns, and uptake and efflux were assessed relative to D-mannitol (extracellular tracer) during a single transit through the column. 2. At tracer concentrations significant unidirectional uptakes were measured for L-leucine (53 +/- 2%), L-phenylalanine (73 +/- 2%), L-serine (40 +/- 4%), L-arginine (42 +/- 3%) and L-ornithine (26 +/- 3%). Uptake for L-proline, D-glucose, dopamine and serotonin was lower (6-10%), whereas uptake for the system A analogue 2-methylaminoisobutyric acid (2-MeAIB) was negligible. Uptakes rapidly decreased with time due to tracer efflux. 3. Endothelial cell transport of L-leucine was markedly inhibited during perfusion with 1 mM-BCH (beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, system L analogue), L-leucine, D-leucine, L-phenylalanine, L-methionine and L-DOPA. 2-MeAIB, L-cysteine, glycine, L-proline, hydroxy-L-proline, L-aspartate and beta-alanine were poor inhibitors, while L-serine and the cationic substrates L-lysine and L-arginine inhibited uptake by 10-35%. 4. When the kinetics of L-leucine transport were examined over a wide range of substrate concentrations (0.025-1 mM) transport was saturable. A single entry site analysis gave a half-maximal saturation constant Kt = 0.24 +/- 0.08 mM (mean +/- S.E.M., n = 5) and a Vmax = 27.8 +/- 4.6 nmol/min per column (approximately 3 x 10(6) cells). 5. Removal of sodium from the perfusate inhibited tracer uptake of L-leucine, L-serine and L-arginine by respectively 20 +/- 5% (n = 3), 77 +/- 5% (n = 3) and 35 +/- 4% (n = 3). 6. Our results provide the first evidence that cultured human endothelial cells of venous origin express a saturable transport system for large neutral amino acids resembling system L described in brain microvascular endothelium. Detection of Na+-dependent and Na+-independent L-arginine uptake is of interest in view of recent reports that this cationic amino acid may be the physiological precursor for nitric oxide released by endothelium.

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Selected References

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  1. Allen L. A., Gerritsen M. E. Regulation of hexose transport in cultured bovine retinal microvessel endothelium by insulin. Exp Eye Res. 1986 Oct;43(4):679–686. doi: 10.1016/s0014-4835(86)80034-3. [DOI] [PubMed] [Google Scholar]
  2. Bassingthwaighte J. B., Sparks H. V. Indicator dilution estimation of capillary endothelial transport. Annu Rev Physiol. 1986;48:321–334. doi: 10.1146/annurev.ph.48.030186.001541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Betz A. L., Gilboe D. D., Drewes L. R. Kinetics of unidirectional leucine transport into brain: effects of isoleucine, valine, and anoxia. Am J Physiol. 1975 Mar;228(3):895–900. doi: 10.1152/ajplegacy.1975.228.3.895. [DOI] [PubMed] [Google Scholar]
  4. Betz A. L., Gilboe D. D., Yudilevich D. L., Drewes L. R. Kinetics of unidirectional glucose transport into the isolated dog brain. Am J Physiol. 1973 Sep;225(3):586–592. doi: 10.1152/ajplegacy.1973.225.3.586. [DOI] [PubMed] [Google Scholar]
  5. Betz A. L., Goldstein G. W. Specialized properties and solute transport in brain capillaries. Annu Rev Physiol. 1986;48:241–250. doi: 10.1146/annurev.ph.48.030186.001325. [DOI] [PubMed] [Google Scholar]
  6. Cancilla P. A., DeBault L. E. Neutral amino acid transport properties of cerebral endothelial cells in vitro. J Neuropathol Exp Neurol. 1983 Mar;42(2):191–199. doi: 10.1097/00005072-198303000-00008. [DOI] [PubMed] [Google Scholar]
  7. Cardelli-Cangiano P., Fiori A., Cangiano C., Barberini F., Allegra P., Peresempio V., Strom R. Isolated brain microvessels as in vitro equivalents of the blood-brain barrier: selective removal by collagenase of the A-system of neutral amino acid transport. J Neurochem. 1987 Dec;49(6):1667–1675. doi: 10.1111/j.1471-4159.1987.tb02424.x. [DOI] [PubMed] [Google Scholar]
  8. Corkey R. F., Corkey B. E., Gimbrone M. A., Jr Hexose transport in normal and SV40-transformed human endothelial cells in culture. J Cell Physiol. 1981 Mar;106(3):425–434. doi: 10.1002/jcp.1041060312. [DOI] [PubMed] [Google Scholar]
  9. Fincham D. A., Mason D. K., Paterson J. Y., Young J. D. Heterogeneity of amino acid transport in horse erythrocytes: a detailed kinetic analysis of inherited transport variation. J Physiol. 1987 Aug;389:385–409. doi: 10.1113/jphysiol.1987.sp016662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gardner M. L., Atkins G. L. Kinetic analysis of transport processes in the intestine and other tissues. Clin Sci (Lond) 1982 Nov;63(5):405–414. doi: 10.1042/cs0630405. [DOI] [PubMed] [Google Scholar]
  11. Gerritsen M. E., Burke T. M., Allen L. A. Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells. Microvasc Res. 1988 Mar;35(2):153–166. doi: 10.1016/0026-2862(88)90059-3. [DOI] [PubMed] [Google Scholar]
  12. Gerritsen M. E., Cheli C. D. Arachidonic acid and prostaglandin endoperoxide metabolism in isolated rabbit and coronary microvessels and isolated and cultivated coronary microvessel endothelial cells. J Clin Invest. 1983 Nov;72(5):1658–1671. doi: 10.1172/JCI111125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gillis C. N. Pharmacological aspects of metabolic processes in the pulmonary microcirculation. Annu Rev Pharmacol Toxicol. 1986;26:183–200. doi: 10.1146/annurev.pa.26.040186.001151. [DOI] [PubMed] [Google Scholar]
  14. Gordon E. L., Pearson J. D., Slakey L. L. The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface. J Biol Chem. 1986 Nov 25;261(33):15496–15507. [PubMed] [Google Scholar]
  15. Jaffe E. A., Nachman R. L., Becker C. G., Minick C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745–2756. doi: 10.1172/JCI107470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. King G. L., Buzney S. M., Kahn C. R., Hetu N., Buchwald S., Macdonald S. G., Rand L. I. Differential responsiveness to insulin of endothelial and support cells from micro- and macrovessels. J Clin Invest. 1983 Apr;71(4):974–979. doi: 10.1172/JCI110852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mann G. E., Peran S. Basolateral amino acid transport systems in the perfused exocrine pancreas: sodium-dependency and kinetic interactions between influx and efflux mechanisms. Biochim Biophys Acta. 1986 Jun 26;858(2):263–274. doi: 10.1016/0005-2736(86)90331-7. [DOI] [PubMed] [Google Scholar]
  18. Mann G. E., Yudilevich D. L. Discrimination of parallel neutral amino acid transport systems in the basolateral membrane of cat salivary epithelium. J Physiol. 1984 Feb;347:111–127. doi: 10.1113/jphysiol.1984.sp015056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Needham L., Cusack N. J., Pearson J. D., Gordon J. L. Characteristics of the P2 purinoceptor that mediates prostacyclin production by pig aortic endothelial cells. Eur J Pharmacol. 1987 Feb 10;134(2):199–209. doi: 10.1016/0014-2999(87)90166-x. [DOI] [PubMed] [Google Scholar]
  20. Norman P. S., Mann G. E. Ionic dependence of amino-acid transport in the exocrine pancreatic epithelium: calcium dependence of insulin action. J Membr Biol. 1987;96(2):153–163. doi: 10.1007/BF01869241. [DOI] [PubMed] [Google Scholar]
  21. Palmer R. M., Ferrige A. G., Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987 Jun 11;327(6122):524–526. doi: 10.1038/327524a0. [DOI] [PubMed] [Google Scholar]
  22. Palmer R. M., Rees D. D., Ashton D. S., Moncada S. L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun. 1988 Jun 30;153(3):1251–1256. doi: 10.1016/s0006-291x(88)81362-7. [DOI] [PubMed] [Google Scholar]
  23. Pardridge W. M. Brain metabolism: a perspective from the blood-brain barrier. Physiol Rev. 1983 Oct;63(4):1481–1535. doi: 10.1152/physrev.1983.63.4.1481. [DOI] [PubMed] [Google Scholar]
  24. Peran S., McGee M. P. Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures. Biochim Biophys Acta. 1986 Apr 14;856(2):231–236. doi: 10.1016/0005-2736(86)90032-5. [DOI] [PubMed] [Google Scholar]
  25. Schmidt H. H., Klein M. M., Niroomand F., Böhme E. Is arginine a physiological precursor of endothelium-derived nitric oxide? Eur J Pharmacol. 1988 Mar 29;148(2):293–295. doi: 10.1016/0014-2999(88)90578-x. [DOI] [PubMed] [Google Scholar]
  26. Shepro D., Dunham B. Endothelial cell metabolism of biogenic amines. Annu Rev Physiol. 1986;48:335–345. doi: 10.1146/annurev.ph.48.030186.002003. [DOI] [PubMed] [Google Scholar]
  27. Smith Q. R., Momma S., Aoyagi M., Rapoport S. I. Kinetics of neutral amino acid transport across the blood-brain barrier. J Neurochem. 1987 Nov;49(5):1651–1658. doi: 10.1111/j.1471-4159.1987.tb01039.x. [DOI] [PubMed] [Google Scholar]
  28. Strum J. M., Junod A. F. Radioautographic demonstration of 5-hydroxytryptamine- 3 H uptake by pulmonary endothelial cells. J Cell Biol. 1972 Sep;54(3):456–467. doi: 10.1083/jcb.54.3.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Syrota A., Girault M., Pocidalo J. J., Yudilevich D. L. Endothelial uptake of amino acids, sugars, lipids, and prostaglandins in rat lung. Am J Physiol. 1982 Jul;243(1):C20–C26. doi: 10.1152/ajpcell.1982.243.1.C20. [DOI] [PubMed] [Google Scholar]
  30. Vinters H. V., Beck D. W., Bready J. V., Maxwell K., Berliner J. A., Hart M. N., Cancilla P. A. Uptake of glucose analogues into cultured cerebral microvessel endothelium. J Neuropathol Exp Neurol. 1985 Sep;44(5):445–458. doi: 10.1097/00005072-198509000-00001. [DOI] [PubMed] [Google Scholar]
  31. Wade L. A., Katzman R. Synthetic amino acids and the nature of L-DOPA transport at the blood-brain barrier. J Neurochem. 1975 Dec;25(6):837–842. doi: 10.1111/j.1471-4159.1975.tb04415.x. [DOI] [PubMed] [Google Scholar]
  32. Yudilevich D. L., Mann G. E. Unidirectional uptake of substrates at the blood side of secretory epithelia: stomach, salivary gland, pancreas. Fed Proc. 1982 Dec;41(14):3045–3053. [PubMed] [Google Scholar]
  33. Zetter B. R. Migration of capillary endothelial cells is stimulated by tumour-derived factors. Nature. 1980 May 1;285(5759):41–43. doi: 10.1038/285041a0. [DOI] [PubMed] [Google Scholar]

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